938 Study of the tumor microenvironment of oral squamous cell carcinoma using multiplex immunofluorescence and image analysis approaches

BackgroundHead and Neck Squamous Cell Carcinoma is the 8th leading cancer worldwide and it is associated with significant morbidity and mortality.1 2 Tumor microenvironment (TME) is dynamic and it plays an important role in head and neck carcinogenesis.3 4 Cytotoxic T-cells, immune checkpoint molecules such as programmed cell death 1 (PD-1), its ligand (PD-L1), and other checkpoints molecules have been described in these tumors.1 3 This study aimed to characterize the TME of oral squamous cell carcinoma (OSCC) and compare with their pathology features.MethodsFour microns thickness consecutives slides from representative OSCC (N=46) cases were stained and analyzed using 11 biomarkers (CK, CD3, CD8, CD68, PD1, PDL1, LAG3, TIM3, ICOS, VISTA, OX40) placed in two multiplex immunofluorescence panels to characterize the TME. For image analysis, the samples were divided in tumor, stroma and peritumoral compartment. Co-expression of markers (cell phenotypes) where analyzed as densities by mm2 in each compartment. For PD-L1 expression by malignant cells (CK+PD-L1+) we set up a cutoff of positive case as ≥ than 1%. Cell phenotypes were correlated with anatomopathological information retrieved from records such as tumor size, margin status, stage and perineural, lymphovascular, and bone invasion among others. Statistical analyses and plots were performed using SPSS and Graphpad prism8 software packages.ResultsWe found significant higher cell density for CK+PDL1+ (P= 0.038), CD3+PDL1+ (P= 0.027), CD3+CD8+PDL1+ (P=0.040) in female patients compared with the male population. Interestingly, smaller tumor size (≤ median, 25mm) showed higher densities of CD3+ (P= 0.006), CD3+CD8+ (P= 0.007), CD3+PDL1+ (P= 0.037), CD3+CD8+PDL1+ (P= 0.016), CD3+ICOS+ (P= 0.036), CD3+VISTA+ (P= 0.001), CD68+ (P= 0.001) and CD68+PD-L1+ (P= 0.008) than large tumors. Additionally, high cell density CD3+OX40+ (P= 0.011) was observed in tumors without margin invasion and high cell density for macrophages CD68+ (p= 0.005) in tumors without bone invasion. In ulcerative and infiltrative tumor pattern we observed higher cell density of CD3+PDL1+ (P= 0.020), CD3+CD8+PDL1+ (P=0.006) and CD3+OX40+ (P= 0.022) than non-ulcerate and no infiltrative pattern. Lastly, 58.7% of cases were PDL1+.ConclusionsOur findings of a diminished immune response in larger tumors might be correlated to their potential role in tumor aggressiveness and progression. Furthermore, high cell density of macrophages on tumor bone invasion may suggest an immune suppressive M2 response supported by the presence of PDL1+ expression. All these results can be the first approach for the development of a treatment based of immune interception.AcknowledgementsThis study was supported by a strategic alliance between the Translational Molecular Pathology-Immunoprofiling las (TMP-IL) at the Department Translational Molecular Pathology, the University of Texas MD Anderson Cancer Center and the Université Claude Bernard Lyon, Centre de Recherche en Cancérologie de Lyon and the Department of Translational Medicine, Centre Léon Bérard, Lyon, France. The authors would acknowledge ITMO Cancer 2020, ”Formation à la Recherche Fondamentale et Translationnelle en Cancérologie” (JB); CLARA 2020 ”Soutien à la mobilité des jeunes chercheurs en oncologie, N° CVPPRCAN000198” (JB); Fondation de France 2020 ”Aide à la mobilité international de médecins et pharmaciens, N° 00112162” (JB); Ligue contre le cancer 2021, comité de Saône-et-Loire (PS); 2017-INCa-DGOS-Inserm_12563: INCa SIRIC-LYriCAN INCa-DGOS-Inserm_12563 (PS)ReferencesCohen EEW, Bell RB, Bifulco CB, Burtness B, Gillison ML, Harrington KJ, et al. The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC). J Immunother Cancer 2019;7(1):184. Bouaoud J, Foy JP, Tortereau A, Michon L, Lavergne V, Gadot N, et al. Early changes in the immune microenvironment of oral potentially malignant disorders reveal an unexpected association of M2 macrophages with oral cancer free survival. Oncoimmunology 2021;10(1):1944554.Mei Z, Huang J, Qiao B, Lam AK. Immune checkpoint pathways in immunotherapy for head and neck squamous cell carcinoma. Int J Oral Sci 2020;12(1):16.Yokota T, Homma A, Kiyota N, Tahara M, Hanai N, Asakage T, et al. Immunotherapy for squamous cell carcinoma of the head and neck. Jpn J Clin Oncol 2020;50(10):1089–96.Ethics ApprovalThe study was conducted in accordance with all applicable laws, rules, and requests of French and European government authorities. Written informed consent was obtained from all patients and the study was approved by the Centre Leon Bérard institutional review board (Lyon, France). Samples were obtained from the CRB Centre Léon Bérard (n°BB-0033-00050) which is quality certified according NFS96-900 French standard and ISO 9001 for clinical trials.